Brett A. Chromy

Selective neuronal degeneration induced by soluble oligomeric amyloid beta protein

The prevailing amyloid hypothesis for Alzheimers disease (AD) holds that amyloid β‐protein (Aβ) causes neuronal degeneration by forming neurotoxic fibrillar structures. Yet, many aspects of AD pathology and symptoms are not well explained by this hypothesis. Here, we present evidence that neurotoxicity of soluble oligomeric Aβ closely corresponds to the selective neurodegeneration so distinctly manifest in AD. Selectivity was first observed in vitro, where only the human central nervous system neuronal cells were susceptible to soluble oligomeric Aβ. Furthermore, in mouse cerebral slice treated with soluble oligomeric Aβ, selective regiospecific toxicity was evident in the hippocampal CA1, a division important for memory, but not in the CA3 subfield. The fibrillar Aβ, however, killed neurons in all regions of the cerebral slice cultures and also in cerebellar slices. Remarkably, even at the highest soluble oligomeric Aβ concentrations, cerebellar neurons were completely spared, consistent with one of the hallmark features of AD pathology. Our observation of the selective neurodegeneration of soluble oligomeric Aβ to neurons involved in cognitive function may provide a new opportunity for the development of an effective AD therapy as well as elucidating the pathological mechanism of AD.

Insertion of Membrane Proteins into Discoidal Membranes Using a Cell-Free Protein Expression Approach

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2.

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimers disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells

Purpose: To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2−/low breast cancers. Experimental Design: HER2-expressing BCSCs (HER2+/CD44+/CD24−/low) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program. Results: Compared with HER2-negative BCSCs, HER2+/CD44+/CD24−/low cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2+/CD44+/CD24−/low cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2–STAT3 network was identified. Conclusion: This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2−/low breast cancer. Clin Cancer Res; 18(24); 6634–47. ©2012 AACR.

Proteomic Characterization of Yersinia pestis Virulence

The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.

Cyclin B1/Cdk1 Coordinates Mitochondrial Respiration for Cell-Cycle G2/M Progression

A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. Here, we show that a fraction of cyclin B1/Cdk1 proteins localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression.

Host–pathogen interactions: a proteomic view

Host–pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host–pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host–pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host–pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.

Efficient maximum likelihood estimator fitting of histograms.

To the Editor: Scientists commonly form histograms of counted events from their data, and extract parameters by fitting to a known model. Anytime a scientist counts photons, molecules, cells or data for individuals in histogram bins and fits that to a distribution, he or she is fitting an event-counting histogram. Here we aim to convince the scientific community to use the maximum likelihood estimator (MLE) for Poisson deviates when fitting event-counting histograms rather than the typically used least-squares measure. We describe how to use the MLE to fit data efficiently and robustly, and provide example code (Supplementary Software). The least-squares measure is the MLE for Gaussian-distributed data. However, event counts in histogram bins are distributed according to the Poisson distribution1. The application of leastsquares procedures to Poisson-distributed data is known to lead to biases, exemplified by results in fitting exponential decays2. The proper procedure with event-counting histograms is to adjust fitting parameters to minimize the MLE for the Poisson distribution. For a dataset x = (x1,x2,...,xN) fitted to a model function f = (f1,f2,... ,fN), the MLE (mle) for Poisson deviates is

Cell-free expression for nanolipoprotein particles: Building a high-throughput membrane protein solubility platform

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.